Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive upper and lower motor neuron degeneration. One of the peculiar clinical characteristics of ALS is the wide distribution in age of onset, which is probably caused by different combinations of intrinsic and exogenous factors. We investigated whether these modifying factors are converging into common pathogenic pathways leading either to an early or a late disease onset. This would imply the identification of phenotypic biomarkers, that can distinguish the two populations of ALS patients, and of relevant pathways to consider in a therapeutic intervention. Toward this aim a differential proteomic analysis was performed in peripheral blood mononuclear cells (PBMC) from a group of 16 ALS patients with an age of onset &le;55 years and a group of 16 ALS patients with an age of onset &ge;75 years, and matched healthy controls. We identified 43 differentially expressed proteins in the two groups of patients. Gene ontology analysis revealed that there was a significant enrichment in annotations associated with protein folding and response to stress. We next validated a selected number of proteins belonging to this functional group in 85 patients and 83 age- and sex-matched healthy controls using immunoassays. The results of the validation study confirmed that there was a decreased level of peptidyl-prolyl cis-trans isomerase A (also known as cyclophilin A), heat shock protein HSP 90-alpha, 78 kDa glucose-regulated protein (also known as BiP) and protein deglycase DJ-1 in PBMC of ALS patients with an early onset. Similar results were obtained in PBMC and spinal cord from two SOD1G93A mouse models with an early and late disease onset. This study suggests that a different ability to upregulate proteins involved in proteostasis, such as foldase and chaperone proteins, may be at the basis of a different susceptibility to ALS, putting forward the development of therapeutic approaches aiming at boosting the protein quality control system.

Figure 2: Validation of candidate phenotypic biomarkers in PBMC of early and late ALS patients. (A–H) PPIA, HSP90, GRP78, ERp57, DJ-1, HSC70, and TDP-43 were analyzed by dot blot immunoassays in PBMC samples from an independent set of ALS patients (n = 85), n = 38 EA and n = 47 LA, and matched controls (n = 83), n = 35 EC and n = 48 LC. Immunoreactivity was normalized to protein loading, as assessed by Ponceau Red staining, and then to the mean values of matched controls (A–F,H). We found that PPIA, HSP90, GRP78, and DJ-1 were significantly lower in early ALS than in late ALS (A–C,E) and that TDP-43 was higher in both early and late ALS compared to matched controls (G). ∗p < 0.05, by Student’s t-test.

Mentions:
In the validation study, we analyzed total protein levels of candidate phenotypic biomarkers, PPIA, HSP90, GRP78, ERp57, and DJ-1, by dot blot analysis in PBMC samples from an independent set of ALS patients (n = 85), n = 38 early ALS and n = 47 late ALS, and matched controls (n = 83), n = 35 early controls and n = 48 late controls (Table 2). Immunoreactivity values for each protein in early and late ALS patients were normalized to the mean values of early and late controls, respectively, similarly to the proteomic analysis. We found that PPIA, HSP90, GRP78, and DJ-1 were significantly lower in early ALS than in late ALS, and therefore can be considered phenotypic biomarkers (Figures 2A–C,E), while ERp57 did not change (Figure 2D). Moreover, PPIA, HSP90, GRP78, and DJ-1 were similarly regulated, with a decreased level in early ALS compared to early controls and an opposite tendency in late ALS (Supplementary Figures S2A–C,E), suggesting that early ALS patients have some defects in the response to stress pathways. In the same validation analysis, we measured another chaperone protein, HSC70, and TDP-43, both not differential in the 2D-DIGE analysis, but up-regulated in PBMC of a cohort of ALS patients with a mean age of onset of 62 ± 10 years, previously reported in a work by our laboratory (Nardo et al., 2011). We found that HSC70 could not distinguish early from late ALS (Figure 2F), but it was up-regulated in late ALS compared to controls (Supplementary Figure S2F). We confirmed that total TDP-43 protein levels in PBMC underscores disease (Figure 2G), in fact it was higher in both early and late ALS compared to controls, but could not discriminate early ALS from late ALS (Figure 2H). It has to be noted that in our assay the total TDP-43 protein level comprise the 43-kDa full-length and fragmented forms (Supplementary Figure S1A).

Figure 2: Validation of candidate phenotypic biomarkers in PBMC of early and late ALS patients. (A–H) PPIA, HSP90, GRP78, ERp57, DJ-1, HSC70, and TDP-43 were analyzed by dot blot immunoassays in PBMC samples from an independent set of ALS patients (n = 85), n = 38 EA and n = 47 LA, and matched controls (n = 83), n = 35 EC and n = 48 LC. Immunoreactivity was normalized to protein loading, as assessed by Ponceau Red staining, and then to the mean values of matched controls (A–F,H). We found that PPIA, HSP90, GRP78, and DJ-1 were significantly lower in early ALS than in late ALS (A–C,E) and that TDP-43 was higher in both early and late ALS compared to matched controls (G). ∗p < 0.05, by Student’s t-test.

Mentions:
In the validation study, we analyzed total protein levels of candidate phenotypic biomarkers, PPIA, HSP90, GRP78, ERp57, and DJ-1, by dot blot analysis in PBMC samples from an independent set of ALS patients (n = 85), n = 38 early ALS and n = 47 late ALS, and matched controls (n = 83), n = 35 early controls and n = 48 late controls (Table 2). Immunoreactivity values for each protein in early and late ALS patients were normalized to the mean values of early and late controls, respectively, similarly to the proteomic analysis. We found that PPIA, HSP90, GRP78, and DJ-1 were significantly lower in early ALS than in late ALS, and therefore can be considered phenotypic biomarkers (Figures 2A–C,E), while ERp57 did not change (Figure 2D). Moreover, PPIA, HSP90, GRP78, and DJ-1 were similarly regulated, with a decreased level in early ALS compared to early controls and an opposite tendency in late ALS (Supplementary Figures S2A–C,E), suggesting that early ALS patients have some defects in the response to stress pathways. In the same validation analysis, we measured another chaperone protein, HSC70, and TDP-43, both not differential in the 2D-DIGE analysis, but up-regulated in PBMC of a cohort of ALS patients with a mean age of onset of 62 ± 10 years, previously reported in a work by our laboratory (Nardo et al., 2011). We found that HSC70 could not distinguish early from late ALS (Figure 2F), but it was up-regulated in late ALS compared to controls (Supplementary Figure S2F). We confirmed that total TDP-43 protein levels in PBMC underscores disease (Figure 2G), in fact it was higher in both early and late ALS compared to controls, but could not discriminate early ALS from late ALS (Figure 2H). It has to be noted that in our assay the total TDP-43 protein level comprise the 43-kDa full-length and fragmented forms (Supplementary Figure S1A).

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive upper and lower motor neuron degeneration. One of the peculiar clinical characteristics of ALS is the wide distribution in age of onset, which is probably caused by different combinations of intrinsic and exogenous factors. We investigated whether these modifying factors are converging into common pathogenic pathways leading either to an early or a late disease onset. This would imply the identification of phenotypic biomarkers, that can distinguish the two populations of ALS patients, and of relevant pathways to consider in a therapeutic intervention. Toward this aim a differential proteomic analysis was performed in peripheral blood mononuclear cells (PBMC) from a group of 16 ALS patients with an age of onset &le;55 years and a group of 16 ALS patients with an age of onset &ge;75 years, and matched healthy controls. We identified 43 differentially expressed proteins in the two groups of patients. Gene ontology analysis revealed that there was a significant enrichment in annotations associated with protein folding and response to stress. We next validated a selected number of proteins belonging to this functional group in 85 patients and 83 age- and sex-matched healthy controls using immunoassays. The results of the validation study confirmed that there was a decreased level of peptidyl-prolyl cis-trans isomerase A (also known as cyclophilin A), heat shock protein HSP 90-alpha, 78 kDa glucose-regulated protein (also known as BiP) and protein deglycase DJ-1 in PBMC of ALS patients with an early onset. Similar results were obtained in PBMC and spinal cord from two SOD1G93A mouse models with an early and late disease onset. This study suggests that a different ability to upregulate proteins involved in proteostasis, such as foldase and chaperone proteins, may be at the basis of a different susceptibility to ALS, putting forward the development of therapeutic approaches aiming at boosting the protein quality control system.